This invention relates to a method for establishing and using a decision point in flow cytometry wherein the decision point defines a point on the axis of a fluorescence histogram for a fluorescent marker of interest such that if the median channel number of cells stained with that fluorescent marker is greater than the decision point then the sample is said to be xe2x80x9cpositivexe2x80x9d for the fluorescence marker used. The invention more particularly relates to a method for using a fluorescent microparticle as a a control to adjust/monitor the decision point in a flow cytometer. The microparticle having a specified fluorescence which corresponds to the point on the fluorescence histogram when used in conjunction with a fluorescently labelled anti-HLA-B27 monoclonal antibody. When a patient sample is tagged with a fluorescently labelled anti-HLA-B27 antibody, if the median fluorescence channel exceeds the decision point, the patient is said to be xe2x80x9cHLA-B27+.xe2x80x9d
Flow cytometry comprises a well known methodology for identifying and distinguishing between different cell types in a non-homogeneous sample. The sample may be drawn from a variety of sources such as blood, lymph, urine, or may be derived from suspensions of cells from solid tissues such as spleen, lymph node or liver. In the flow cytometer, cells are passed substantially one at a time through one or more sensing regions wherein each cell is illuminated by an energy source. The energy source generally comprises means that emits light of a single wavelength in a sensing region such as that provided by a laser (e.g., He/Ne or argon) or a mercury arc lamp with appropriate bandpass filters. Different sensing regions can include energy sources that emit light at different wavelengths.
In series with each sensing region, various light collection means, such as photomultiplier tubes, are used to gather light that is refracted by each cell (generally referred to as forward light scatter), light that is reflected orthogonal to the direction of the flow of the cells through a sensing region (generally referred to as orthogonal or side light scatter) and one or more light collection means to collect fluorescent light that may be emitted from the cell, if it has been tagged with one or more fluorescent markers, as it passes through a sensing region and is illuminated by the energy source. Light scatter is generally correlated with physical characteristics of each cell such as size and granularity.
Flow cytometers further comprise data recording and storage means, such as a computer, wherein separate channels record and store the light scattered and fluorescence emitted by each cell as it passes through a sensing region (i.e., the data collected for each cell comprises a xe2x80x9crecorded eventxe2x80x9d). The recorded events then can be displayed by plotting orthogonal light scatter versus forward light scatter in either real time or by reanalysis of the data after the events have been recorded. U.S. Pat. Nos. 4,599,307 and 4,727,020 describe of the various components that comprise a flow cytometer and also the general principles of its use.
Monoclonal antibodies are particularly useful in flow cytometry when conjugated, directly or indirectly, to fluorescent dyes (generically this combination is referred to as an xe2x80x9cimmunofluorescent markerxe2x80x9d). Monoclonal antibodies have been made against a large number of antigens present on and within cells. Such antibodies are used to identify certain populations of hematopoietic cells as well as subpopulations thereof. An immunofluorescent marker is added to a sample of cells and the sample then is analyzed by means of flow cytometry. The light emitted by the fluorescent dye as it is excited by the energy source is stored and recorded as described above. PCT Appl. No. PCT/CA92/00105 describes the extent to which monoclonal antibodies have been and are being used to identify various cell populations and subpopulations.
When a recorded event includes fluorescent data, the data can be displayed in a fluorescence histogram such as is shown in FIG. 2 of U.S. Pat. No. 4,727,020. If multiple immunofluorescent markers are used (wherein each dye has an emission spectra that is distinguishable from the other dyes used), the data for a recorded event can be displayed in a multi-dimensional format such as shown in FIG. 3 of the same patent.
Looking more closely at FIG. 2a in that patent, it can be seen that not all cells express the same amount of fluorescence. This may be due to reaction conditions or may be due to differences between the levels of expression of the antigen on the cell. It also can be seen that there are a large number of cells that have little or no fluorescence intensity. Generally, these cells would be referred to as being xe2x80x9cnegativexe2x80x9d for expression of the immunofluorescent marker while the remaining cells would be referred to as being xe2x80x9cpositivexe2x80x9d for the immunofluorescent marker.
Looking at FIG. 3, one can distinguish between cells on the x and y axes that are clearly xe2x80x9cpositivexe2x80x9d (i.e., they are far away from the origin), and cells in the bottom left corner of the figure that are xe2x80x9cnegativexe2x80x9d (i.e., they are close to the origin). For both FIG. 2 and FIG. 3, however, it is not immediately apparent from the displays which cells should be characterized definitively as belonging in either class nor is it apparent that one would classify the individual from whom this sample was taken as being positive or negative for the markers used.
Because the level of fluorescence intensity for any given population of tagged cells in a sample is not always clear, many methods have been developed to try to separate xe2x80x9cpositivexe2x80x9d and xe2x80x9cnegativexe2x80x9d cells. U.S. Pat. No. 4,987,086 describes a gating method which can be used to set boundaries or xe2x80x9cgatesxe2x80x9d in a two dimensional display in order to distinguish between such cells. The method makes use of scatter parameters and fluorescent parameters to arrive at a decision point for each fluorescent marker as to the boundary between positive and negative cells.
The information provided by the method described in U.S. Pat. No. 4,987,086, however, relates only to a calculation of the number of cells present in one or more cell populations or subpopulations for the individual sample being examined. It does not give an indication whether a particular sample can be said to be xe2x80x9cpositivexe2x80x9d or xe2x80x9cnegativexe2x80x9d for the a particular marker when that marker is viewed in the context of a population of patients. For example, in certain diseases, the presence or absence of a particular marker may be indicative of a disease state or of susceptibility to such disease. In a sample taken from a patient, there may be individual cells that are xe2x80x9cpositivexe2x80x9d or xe2x80x9cnegativexe2x80x9d on a fluorescence plot of those cells. The plot will have a median fluorescence channel. Unless the channel number for that patient""s sample is compared with a decision point derived from a population of patients, the total number of cells which are positive or negative does not make full use of the information present. Thus, merely classifying each cell as positive or negative for a marker is not enough.
The present invention comprises a method to identify and set a decision point in order to discriminate between a positive and negative sample of cells wherein those cells have been tagged with a fluorescent marker. The decision point comprises a decision point on a fluorescence axis. The decision point is set so that it corresponds to the fluorescence channel at the desired sensitivity and specificity for the fluorescent marker. This is done by analysis of known patient samples. In a preferred embodiment, the desired sensitivity is 100% and the desired specificity is at least 97%. If a patient sample that has been tagged with a fluorescent marker which is specific for the cell marker of interest has a median channel number greater than the decision point, the sample is said to be xe2x80x9cpositivexe2x80x9d for that marker.
The decision point may be set in software used in conjunction with the flow cytometer, wherein the software calculates the median channel number for all the recorded events and compares that to the decision point for that marker. Alternatively, or in addition, a microparticle having fluorescence characteristics similar to the fluorescent marker can be used. In this embodiment, the particle is used to adjust/monitor the instrument so that if the decision point were set at xe2x80x9c166xe2x80x9d, the particles can be run on the instrument prior to or at the same time as the fluorescent marker to be sure that the instrument reads 166 as 166.
After the decision point is set, one or more fluorescent markers are added to a sample containing cells. The cells then are run through a flow cytometer as described above. The median fluorescence channel then is determined for the cells tagged with each fluorescence marker. If the median channel number exceeds the decision point for that marker, the population of cells is considered xe2x80x9cpositivexe2x80x9d for expression of that marker.
It should be apparent that multiple fluorescent markers can be used in order to identify or limit the population or subpopulation of cells being analyzed. In this embodiment, it is possible to use combinations of immunofluorescent markers alone in order to identify and isolate certain subpopulations of cells and/or in combination with one or more nucleic acid stains for the same purpose. It further is to be appreciated that multiple decision points can be used in multi-dimensional analysis of data wherein a separate decision point is used in conjunction with each fluorescent marker.
This invention has particular utility in the analysis of blood cells from patients suspected of having certain diseases such as ankylosing spondylitis (xe2x80x9cASxe2x80x9d). It is known that certain individuals who are characterized as being HLA-B27+ are more likely to develop such diseases. Thus, typing the individual for HLA type is an important diagnostic tool.
In this embodiment, a fluorescently labelled anti-CD3 monoclonal antibody and a fluorescently labelled anti-HLA-B27 monoclonal antibody are prepared. The fluorescence:protein ratio for the HLA-B27 antibody is determined. It is important that this ratio not vary significantly because the fluorescence intensity of the antibody will effect the median channel number (i.e., if there are fewer fluors on an antibody, then the median channel number of the cells tagged with those antibodies will be lower and therefore could give a potentially false result). A microparticle having similar fluorescence properties as the labelled HLA-B27 antibody also is prepared.
The microparticle is run on the instrument in order to assure that the instrument will read the desired decision point in the proper fluorescence channel. The labelled antibodies then are added to blood. The fluorescently labelled anti-CD3 monoclonal antibody is used in conjunction with forward scatter to gate on only those cells that are positive for this antibody. The median fluorescence channel of the HLA-B27+ T cells falling within the gates then is determined. If the median channel number exceeds the decision marker channel number, the sample is said to be xe2x80x9cpositive.xe2x80x9d